This invention relates to a chemiluminescent reagent capable of giving controlled chemiluminescence, useful for, e.g., detection or quantitative analysis of a variety of materials by the chemiluminescent analysis. This invention also relates to a method for measuring peroxidase activity in the presence of a hydrogen acceptor by the chemiluminescent analysis using the above chemiluminescent reagent, and chemiluminescent enzyme immunoassay using peroxidase as the marker.
Lucigenin (N,Nxe2x80x2-dimethyl-9,9xe2x80x2-bisacridinium dinitrate), which has been widely used for a long time for a variety of microanalysis methods as a chemiluminescent reagent, is known to generate light slightly in an aqueous alkaline solution and strongly in the presence of hydrogen peroxide. It can be used to quantitatively analyze hydrogen peroxide, because it emits light quantitatively in the presence of hydrogen peroxide and an alkali, but is difficult to apply to chemiluminescent enzyme immunoassay (hereinafter referred to as CLEIA, as necessary), which measures enzyme activity by extent of luminescence it produces by its reaction while controlling the luminescence. One of the methods proposed to solve these problems uses glucose oxidase as the marker enzyme to produce chemiluminescence, extent of which depends on concentration of the material to be analyzed, by acting hydrogen peroxide, as the product of glucose oxidation, on lucigenin in the presence of an alkali.
However, the CLEIA method with glucose oxidase as the marker involves problems of time-consuming reagent preparation and handling of the luminescent system. Luminol is used as the chemiluminescent reagent applicable to the CLEIA method with peroxidase, which can be handled relatively easily, as the marker. However, it cannot always show sufficient sensitivity, even in the presence of luminescent promoter, e.g., p-iodophenol.
Therefore, there are increasing demands for chemiluminescent reagents which can be prepared easily, simplify the luminescent analysis procedure, are applicable to the CLEIA method with peroxidase as the marker, and realize high-sensitivity analysis.
It is also necessary to further improve sensitivity of the CLEIA method with peroxidase as the marker, because it is required to quantitatively cover increasingly lower concentrations of the materials to be analyzed.
It is an object of the present invention to provide a novel chemiluminescent reagent for chemiluminescence appearing depending on molar concentration of peroxidase with the substrate of a peroxide, e.g., hydrogen peroxide, as the hydrogen acceptor.
It is another object of the present invention to provide a chemiluminescent reagent which produces a high extent of chemiluminescence and is highly stable.
It is still another object of the present invention to provide a novel analysis based on the chemiluminescence which uses the above chemiluminescent reagent to measure peroxidase activity at higher sensitivity.
It is still another object of the present invention to provide a novel chemiluminescent enzyme immunoassay by the new immunoassay system, developed on the basis of the chemiluminescence which uses the above chemiluminescent reagent and peroxidase enzyme as the marker to measure a material to be analyzed at higher sensitivity.
The inventors of the present invention have found, after having extensively studied to achieve the above objects, that a chemiluminescent reagent which contains a charge-transferring complex of N,Nxe2x80x2-disubstituted-9,9xe2x80x2-bisacridinium salt and N,N-disubstituted carboxylic amide compound,
a chemiluminescent reagent obtained by incorporating an aminoalcohol compound into the above chemiluminescent regent,
a chemiluminescent reagent prepared by reacting an N,Nxe2x80x2-disubstituted-9,9xe2x80x2-bisacridinium salt with N,N-disubstituted carboxylic amide compound while being irradiated with light, or
a chemiluminescent reagent prepared by reacting an N,Nxe2x80x2-disubstituted-9,9xe2x80x2-bisacridinium salt with N,N-disubstituted carboxylic amide compound while being irradiated with light, wherein an aminoalcohol compound is added to the reaction system during and/or after the charge-transfer reaction
do not react with hydrogen peroxide at a specific pH level but produce chemiluminescence in the simultaneous presence of hydrogen peroxide and peroxidase to an extent determined by molar concentration of the peroxidase, reaching the present invention.
First, the present invention relates to a chemiluminescent reagent (hereinafter referred to as Chemiluminescent Reagent I, as necessary) characterized by the chemiluminescence produced in the presence of a peroxide, extent of which varies depending on concentration of peroxidase enzyme, which comprises, as the major ingredients, a charge-transferring complex of N,Nxe2x80x2-disubstituted-9,9xe2x80x2-bisacridinium salt, shown by the general formula (1): 
(wherein, R1 and R2 are each selected from the group consisting of an alkyl, aryl and halogenated aryl groups, and may be the same or different; R3, R4, R5 and R6 are each selected from the group consisting of hydrogen, an alkyl, aryl, alkoxy and aryloxy groups and haolgen, and may be the same or different; and X. is an acid radical as the residue left by the electron transferring from the counter anion of the bisacridinium salt as the precursor), and an N,N-disubstituted carboxylic amide compound shown by the general formula (2): 
(wherein, R1 is selected from the group consisting of hydrogen, an alkyl group having a carbon number of 1 to 10, an alkenyl group having a carbon number of 2 to 10 and an aryl group having a carbon number of 6 to 20, wherein the aryl group may be substituted with an alkyl, nitro, hydroxyl or amino groups, halogen or the like; R2 is selected from the group consisting of methyl and ethyl groups; and R3 is selected from the group consisting of an alkyl group having a carbon number of 1 to 10, an alkenyl group having a carbon number of 2 to 10 and an aryl group having a carbon number of 6 to 20, wherein the aryl group may be substituted with an alkyl, nitro, hydroxyl or amino groups, halogen or the like, and R1 and R3 may be bonded to each other to form a ring together with the carbon atom and nitrogen atom which are in the carbonyl and amide groups, respectively, to which each of R1 and R3 are bonded).
Second, the present invention relates to a chemluminescent reagent (hereinafter referred to as Chemiluminescent Reagent II, as necessary) characterized by the chemiluminescence produced in the presence of a peroxide, extent of which varies depending on concentration of peroxidase enzyme, which comprises, as the major ingredients, a charge-transferring complex of N,Nxe2x80x2-disubstituted-9,9xe2x80x2-bisacridinium salt shown by the general formula (1), N,N-disubstituted carboxylic amide compound shown by the general formula (2) and aminoalcohol compound shown by the general formula (3)
(HOR)mNH3xe2x88x92mxe2x80x83xe2x80x83(3)
(wherein, R is a divalent aliphatic hydrocarbon group having a carbon number of 1 to 5; and (m) is an integer of 1 to 3).
Third, the present invention relates to a chemluminescent reagent (hereinafter referred to as Chemiluminescent Reagent III, as necessary) prepared by reacting, in the presence of irradiated light, an N,Nxe2x80x2-disubstituted-9,9xe2x80x2-bisacridinium salt shown by the general formula (1A): 
(wherein, R1 and R2 are each selected from the group consisting of an alkyl, aryl and halogenated aryl groups, and may be the same or different; R3, R4, R5 and R6 are each selected from the group consisting of hydrogen, an alkyl, aryl, alkoxy and aryloxy groups and haolgen, and may be the same or different; and Xnxe2x88x92 is an n-valent anion; and (n) is 1 or 2) with an N,N-disubstituted carboxylic amide compound shown by the general formula (2): 
(wherein, R1 is selected from the group consisting of hydrogen, an alkyl group having a carbon number of 1 to 10, an alkenyl group having a carbon number of 2 to 10 and an aryl group having a carbon number of 6 to 20, wherein the aryl group may be substituted with an alkyl, nitro, hydroxyl or amino groups, halogen or the like; R2 is selected from the group consisting of methyl and ethyl groups; and R3 is selected from the group consisting of an alkyl group having a carbon number of 1 to 10, an alkenyl group having a carbon number of 2 to 10 and an aryl group having a carbon number of 6 to 20, wherein the aryl group may be substituted with an alkyl, nitro, hydroxyl or amino group, halogen or the like, and R1 and R3 may be bonded to each other to form a ring together with the carbon atom and nitrogen atom which are in the carbonyl and amide groups, respectively, to which each of R1 and R3 are bonded).
Fourth, the present invention relates to a chemluminescent reagent (hereinafter referred to as Chemiluminescent Reagent IV, as necessary) prepared by reacting an N,Nxe2x80x2-disubstituted-9,9xe2x80x2-bisacridinium salt shown by the general formula (1A) with an N,N-disubstituted carboxylic amide compound shown by the general formula (2) while being irradiated with light, wherein an aminoalcohol compound shown by the general formula (3)
xe2x80x83(HOR)mNH3xe2x88x92mxe2x80x83xe2x80x83(3)
(wherein, R is a divalent aliphatic hydrocarbon group having a carbon number of 1 to 5; and (m) is an integer of 1 to 3).is added to the reaction system during and/or after the charge-transfer reaction.
Fifth, the present invention relates to a method for measuring peroxidase activity in the presence of a hydrogen acceptor by the chemiluminescent analysis using one of the above chemiluminescent reagents of the present invention.
Sixth, the present invention relates to a chemiluminescent enzyme immunoassay, which comprises mixing an antibody or antigen marked with peroxidase enzyme with an antibody, antigen or agglomerate thereof in a sample to be analyzed to form the immune complex from the marker/antigen-antibody complex by the antigen-antibody reaction, separating the immune complex, producing its chemiluminescence in the presence of a hydrogen acceptor by the aid of the above chemiluminescent reagent, and measuring the luminescence intensity to quantitatively analyze the anti-gen or antibody in the sample.